Downregulation of CD40L–CD40 attenuates seizure susceptibility and severity of seizures

Unregulated neuro-inflammation mediates seizures in temporal lobe epilepsy (TLE). Our aim was to determine the effect of CD40–CD40L activation in experimental seizures. CD40 deficient mice (CD40KO) and control mice (wild type, WT) received pentenyltetrazole (PTZ) or pilocarpine to evaluate seizures and status epilepticus (SE) respectively. In mice, anti-CD40L antibody was administered intranasally before PTZ. Brain samples from human TLE and post-seizure mice were processed to determine CD40–CD40L expression using histological and molecular techniques. CD40 expression was higher in hippocampus from human TLE and in cortical neurons and hippocampal neural terminals after experimental seizures. CD40–CD40L levels increased after seizures in the hippocampus and in the cortex. After SE, CD40L/CD40 levels increased in cortex and showed an upward trend in the hippocampus. CD40KO mice demonstrated reduction in seizure severity and in latency compared to WT mice. Anti-CD40L antibody limited seizure susceptibility and seizure severity. CD40L–CD40 interaction can serve as a target for an immuno-therapy for TLE.

www.nature.com/scientificreports/ Also, we analyzed the effects of intranasal anti-CD40L antibody treatment against PTZ-induced seizures. We observed that either genetic deficiency of CD40 or intranasal administration of anti-CD40L antibody was able to limit seizure susceptibility, reducing the frequency of induced acute seizures. Therefore, targeting CD40-CD40L or their molecular signaling pathways could pave the way toward a new therapeutic approach against epilepsy.

Materials and methods
Studies were performed according to the National Institutes of Health (NIH) guidelines and in accordance with nationally accepted principles in the care and use of experimental animals. The Institutional Animal Care and Use Committee at Eastern Virginia Medical School approved the animal protocol for this study (IACUC, #17-012). Water and food were available for ad libitum consumption in individual cages located at the EVMS Comparative Medicine Vivarium. Animals were fed a diet from ENVIGO containing a nutrient profile with 6.2% fat and 18.6% protein. All efforts were made to minimize pain and suffering and to reduce the number of mice used in these experiments. For euthanasia, animals were deeply exposed to isoflurane.
Mice. Adult male mice (28-33 g) used included CD40 receptor-deficient knockout (B6.129P2-Tnfrsf5 tm1kitk, The Jackson Laboratory), and C57BL/6 as a control (WT) from The Jackson Laboratory. CD40KO mice were backcrossed to B6, and bought at ~ 13 weeks from Jackson Laboratories and used after arrival for experiments. For seizure susceptibility, each mouse received injections every 5 min until the onset of the first retropulsive myoclonus, defined as a myoclonic jerk resulting in backward movement of the head and shoulders, and then subsequently euthanized. The latency to the onset of myoclonic jerk was the primary metric recorded. Seizures were classified according to the Racine scale 6 . (0: normal behavior-walking, exploring, sniffing, grooming; 1: immobile, staring, jumpy, curled-up posture; 2: automatisms-repetitive blinking, chewing, head bobbing, vibrissae twitching, scratching, face washing, "star gazing"; 3: partial-body clonus, occasional myoclonic jerks, shivering; 4: whole-body tonic-clonus, "corkscrew" turning and flipping, loss of posture, rearing, falling; 5: non-intermittent tonic-clonic seizure activity). Mice were observed continuously for at least 2 h with data recorded regarding time to achievement of respective Racine stage and duration of seizures. were rinsed in water, dehydrated with ethanol, placed in xylene, mounted, and coverslipped. Cortex, dentate gyrus, CA1, and CA3 regions from the right hippocampus were examined by using standard light microscopy with a Zeiss imaging microscope system. Using a Leica CM1950 Cryostat, 20 micron-thick sections were cut and dried overnight. After slides were dry, they were rehydrated using Xylene and ethanol baths. Slides were treated with serum and other protein complexes to block nonspecific binding and were incubated overnight with multiple primary antibodies. Slides were washed with PBST and were incubated with a Horseradish Peroxidase conjugated secondary antibody. After at least an hour of the secondary incubation, slides were washed with PBST and were stained with 3,3′-Diaminobenzidine which developed the cell type isolated with the primary antibody. Imaging was performed using an Olympus microscope.

Immunohistology.
Western blotting and ELISA. Samples that were snap frozen after dissection were placed in a -80 °C refrigerator until Western blotting was performed. To prepare the lysates, about 300 mg of tissue (cortex and hippocampus) was placed in 500uL of RIPA buffer. A homogenizer was used on the samples multiple times until the tissue disintegrated as much as possible. Samples were then agitated in an orbital shaker for 2 h at 4 °C. Samples were homogenized once more and then placed in a centrifuge at 4 °C and set to 12,000RPM for 20 min. www.nature.com/scientificreports/ using 100 V over an hour. Nitrocellulose membranes after transfer were then hybridized with the primary antibody in a blocking solution that contained BSA and TBST, set overnight. Primary antibodies included CD40 (abcam.com, ab13545: 1:1000 concentration), CD40L (abcam.com, ab52750: 1:500 concentration), P38 (Invitrogen, PA5-17713: 1:1000 concentration), PP38 (Invitrogen, MA5-15177: 1:1000 concentration), and B-actin (Biolegend, Catalog # 622102: 1:1000 concentration). Membranes were then washed three times prior to secondary antibody hybridization and three more times after. Imaging was performed using the LicorOdyssey system (Li-Cor, State, USA). ELISA was followed by manufacture instructions (CD40L TNFSF5 ab119517, abcam.com).
Synaptosome extraction. Brains were removed after euthanasia from control animals (saline IP) and from those with tonic-clonic seizures (Racine's score > 3) following PTZ administration (75 mg/kg IP); then cortical and hippocampal regions were microdissected, and snap frozen at -80C. From those samples, synaptosomes were extracted using a tissue homogenizer and the Syn-PER Synaptic Protein Extraction Reagent (Thermo-Fisher Scientific Catalog # 87793) according to manufacturer's protocol to extract synaptosome fractions from samples and controls. After the completion of the last centrifugal step, synaptosome pellets were re-suspended in the Syn-PER Synaptic Protein Extraction Reagent at around a concentration of 1 ml per 400 mg of tissue. Then CD40L and CD40 concentration was measured using a CD40L ELISA (ab119517, abcam.com) kit and a CD40 ELISA (ab100674, abcam.com) kit respectively. A separate protein curve was generated for each ELISA, and the respective protein concentrations of each sample and control was found through the plot of the respective protein curve.
Status epilepticus. Study mice were pretreated with scopolamine injections intraperitoneally (IP) (1 mg/ kg, IP), 30 min prior to status epilepticus induction. Subsequently, Pilocarpine Hydrochloride (280 mg/kg, IP, Sigma Aldrich) was injected and mice were observed over a period of four hours to ensure normal health status. Control mice were injected with equal amounts of sterile saline intraperitoneal. During the first post-pilocarpine observational period (2-4 h after Pilocarpine), mice were evaluated to assess the development of the status epilepticus using the Racine scale 6 . Racine stage equal or above 4 was considered as seizure severity. The mice recovered at least 2 h post-pilocarpine and midazolam (8 mg/kg, IP) was administered as provided by the veterinarian staff at EVMS. Control mice received scopolamine and sterile saline (sham). Mice were left in their appropriate acrylic cages with feed and water over the following 24 h. Mice were monitored every 8 h during the 24-h observational period to ensure healthy levels of hydration and activity.
AntiCD40L administration. The InVivoMab Anti-Mouse CD40L (CD154) antibody blocks CD40-CD40L interaction in vivo as previously reported in various articles 23,24 . The molecule was validated by BioXCell. For intranasal administration, the InVivoMabAnti-MouseCD40L was diluted to a concentration of 2 mg/mL. The initial concentration was 6.69 mg/mL, approximately 300 µl of antibody solution was diluted into 700 µl of sterile saline. The solution was stored in an Eppendorf tube and kept at 4C until it was to be used. At the time of the experiment, 5 µl of the 2 mg/mL CD40L in sterile saline solution was administered to each naris. Animals were administered InVivoMab Anti-Mouse CD40L (CD154) antibody 2 h before PTZ administration.
Statistical analysis. CD40 positive cells of different morphologies per field (40X) were semi-quantified.
Statistical comparisons were conducted among two groups and standard errors of the mean (SEM) by Student's t-test for statistical significance (p < 0.05). Seizures after pilocarpine and percentage of seizure severity was analyzed using non-parametric Wilcoxon signed rank test for statistical significance (p < 0.05). All data analyses and graphics were performed using JMP, Version 8, statistical discovery from SAS, www. jmp. com, Cary, NC, 1989-2021.

CD40 is expressed in a population of human TLE.
A public human tissue database shows that the CD40IR is not detected in normal neural tissue (https:// www. prote inatl as. org/ ENSG0 00001 01017-CD40/ tissue). CD40L-CD40 has not been reported in human epilepsy at the time that this manuscript was submitted. Using an immunohistological approach in hippocampal cryosections obtained from patients (n = 4) that underwent neurosurgical treatment for Temporal Lobe Epilepsy (TLE), the CD40 immunoreactivity (IR) was highly expressed in hippocampal regions (Fig. 1). The CD40 IR labeled cells (Fig. 1D,E) resemble astrocytes and neurons 25,26 . Since no current literature or database shows CD40-CD40L expression in normal human neural tissue, the finding of CD40-CD40L in TLE patients was significant but not further analyzed, other than this descriptive analysis finding.
CD40-CD40L increased after seizures. CD40 is expressed in neurons in the brain from the neocortex and hippocampus in adult mice 27 . However, the hippocampal molecular expression of CD40 is relatively low compared to CD40L 15 . To determine the presence of CD40 in neural terminals by using immunohistological techniques, we observed that the distribution of immunoreactivity (IR) against CD40 presented a trend to be expressed more densely in post-synaptic terminals (Fig. 2C, Supplementary Fig. 2) than pre-synaptic terminals of naive adult mice. Following tonic-clonic seizures (Racine's score > 3) induced by systemic administration of PTZ, CD40 IR was highly expressed mainly in the neurons located in the somatosensory cortex and as a fibrillar pattern in the hippocampus ( Fig. 2A, Supplementary Fig. 2). Moreover, CD40 IR was remarkably increased mainly in CA3 hippocampal subregions and cortex after those seizures compared to control (p < 0.05) (Fig. 2B). The concentration of CD40 in synaptosome fractions significantly increased in pooled cortical and hippocam-  (Fig. 2D). However, CD40L in synaptosome fractions did not show a statistically significant difference (data not shown). In addition, CD40 IR was associated with an increase in

CD40L-CD40 concentration was higher 24 h after status epilepticus. Neuro-inflammation plays
a critical role in the development of epilepsy during the acute phase of epileptogenesis, approximately 24 h after SE in the pilocarpine model of TLE 6,9,10 . Using an enzyme-linked immunosorbent assay (ELISA), we observed an increase in both CD40L and CD40 after SE. Consistent with this previous observation, using a Western Blot, it was seen that CD40 increases in the cortex and the hippocampus (Fig. 3B). Considering that CD40L-CD40 interaction is key in activating an inflammatory process, we evaluated the relationship of the concentration of CD40L-CD40 in the brain by analyzing an index between CD40L over CD40 concentrations. We observed that CD40L/CD40 increased relatively more than CD40 in cortex (Control  (Fig. 3B). Additionally, since the p38MAPK participates in CD40 signaling pathway and has been implicated in epilepsy via a c-Jun N-terminal kinase 28 , we studied the phosphorylation of p38 after SE 28,29 . During the acute phase of epileptogenesis, the relationship between pp38 and p38 increased in cortex (Control: mean: 0.24 ± 0. CD40 deficiency attenuated seizure susceptibility. Given the lack of data concerning the role of CD40 during seizures, we aimed to determine if CD40 deficiency (i.e.CD40KO) affects seizure susceptibility and/or severity. Successive sub-convulsive doses of PTZ (10 mg/kg) were administered to determine the threshold for different types of seizures using Racine's score 6,25 . CD40KO (n = 7) mice exhibited low mortality (Fig. 4A) and reduced seizure occurrence compared to WT (n = 7), with statistically significant differences at  www.nature.com/scientificreports/ CD40 deficiency limited severity of SE. Since the deficiency of CD40 limited seizure susceptibility (Fig. 4), we inquired whether CD40 deficiency was sufficient to mitigate the severity of status epilepticus. We observed that CD40KO mice exhibited seizures with reduced severity compared to WT (Racine's score in CD40KO: 2.8 ± 0.2 S.E.M., n = 10 vs. WT: 3.42 ± 0.18 S.E.M., n = 21; p = 0.01, t = 2.4) (Fig. 4D). Also, CD40 deficiency prevented mortality following SE. (CD40KO: 100% survival vs. WT: 80%, p = 0.04, Chisquare = 2.1) (Fig. 4E).

Discussion
We observed that CD40 is expressed in human TLE, upregulated after seizures in hippocampal regions, and corresponds with an upregulation of CD40L. In mice, CD40L/CD40 increases 24 h after SE in the cortex, as well as in the hippocampus. A decrease in CD40, whether through CD40-deficient mice or by blocking CD40 interactions using in vivo anti-CD40L antibody administration, limits PTZ-induced seizure severity. CD40 is expressed in 50% of patients with TLE. Due to the lack of CD40 research in the normal brain (https:// www. prote inatl as. org/ ENSG0 00001 01017-CD40/ tissue), we hypothesize that CD40 could play a role in seizure severity or frequency caused by an unresolved neuro-inflammatory process in those patients. We plan to continue to explore this relationship in the future. www.nature.com/scientificreports/ It is well-known that CD40 is seen in post-injury inflammation and can be used as a biomarker. This raises the question of the role of CD40 in epileptogenesis 33 . Levels of CD40 were increased predominantly in neural terminals in the hippocampus and cortex following induction of seizures. An increase in CD40L was also seen in the hippocampus and cortex, 24 h after status epilepticus, indicating a possible sustained response. However, additional studies are needed to verify the CD40L-CD40 post-seizure time course, its relationship to various brain regions, and cellular population response. The results of these findings will determine if levels of CD40L-CD40 is an acute response, sustained after seizures, or spontaneously resolved to physiological levels after seizure culmination.
The CD40KO mice, demonstrated a statistically significant reduction in seizure susceptibility, suggesting that CD40-CD40L may be directly involved in mediating ictogenesis or as a mechanism of seizure propagation. The CD40KO mice that developed seizures had a statistically significant decrease in seizure severity with no mortality, suggesting that CD40 mediates a mechanism with neuronal hyper-excitability. These observations warrant further exploration of the role of CD40 neurotransmission impairments in epilepsy in depth. Recently, studies show that postnatal CD40-deficient mice present with a reduction of excitatory hippocampal pyramidal terminals neurons compared to wild type littermate mice. At this location, dendrite arbors of inhibitory striatal medium spiny neurons are increased in size and more branched in the absence of CD40 15 . Additional research is required to evaluate how CD40 mediates the formation of aberrant terminals in epileptogenesis 6 . Anti-CD40 attenuates the release of these cytokines and the resultant blood-brain barrier (BBB) dysfunction 34 . Several studies have implicated CD40-CD40L interaction in the upregulation of endothelium adhesion molecules and leukocyte transmigration, which may influence the integrity of the BBB 26,27 . Further BBB integrity and PTZ pharmacodynamics studies might be useful in our understanding of this scenario.
Our data show that intranasal administration of in vivo anti-CD40L antibody is effective to limit acute seizures (Fig. 5). Anti-CD40L antibodies are currently one of the leading new treatments still under investigation in patients with cancer due to their ability to modulate immune responses. The mechanism of anti-CD40L antibodies in cancer therapy is to enhance the immune system through the downstream effects of CD40 activation and marking malignant cells for destruction 35 . Limited analysis has been conducted on anti-CD40 as a receptor antagonist/blocker. In this condition, our approach could be useful to explore a GBM induced seizure.
In addition, the CD40 molecular signaling may play a role in the development of epilepsy and drug-resistant epilepsy through the activation of P38 MAPK. Our data reveals an increase in phosphorylated p38 in our mouse models with epilepsy. This is consistent with previously published literature showing that phosphorylation of p38MAPK may contribute to epileptogenesis either directly or through downstream effects 27 .
CD40 is commonly expressed on immune cells such as monocytes and dendritic cells and non-immunological cells such as neurons, microglia, and endothelial cells 33,36,37 . Additionally, CD40 contributes to the post-injury inflammatory environment 33 . Microglial activation by lipopolysaccharides has been shown to increase the expression of CD40 and CD40L secretion, in turn increasing the secretion of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 34 .
On the other hand, CD40 could mimic or act synergistically with the function of the pro-inflammatory cytokine TNF-α which plays a role in acute seizures 38 . TNF-α, released from physiologically activated microglia and astrocytes, contributes to the homeostatic level of glutamate via TNF receptor 1 (TNFR1) and regulates the formation and organization of excitatory and inhibitory synapses [39][40][41] . Following an injury, TNF-α up-regulates AMPA receptors, augmenting glutamatergic transmission causing neurotoxicity and hyper-excitability exacerbated by induction of GABA receptor endocytosis, which reduces the inhibitory drive. Since TNF signaling may have an important role not only in ictogenesis, but also in early phases of epileptogenesis, anti-TNF-α therapy for epilepsy is also under debate due to the suspected risks of demyelination, infection, and cancer development.
Although there is an increase in CD40L in epilepsy, its role in TLE has not been fully elucidated, and the present study suggests that CD40L-CD40 interaction could be a promising target for early therapeutic intervention in TLE and could prevent the onset of TLE. This approach could also be expanded to neurological disorders that similarly involve disruption of neuronal networks, such as Alzheimer's disease. The results described in this study highlight the involvement of the CD40-CD40L pathway in the development of epilepsy. This provides the groundwork for potential exploration of CD40-CD40L as a molecular target for the prevention and treatment of epilepsy.